Lipofectamine™ 3000 Reagent
Lipofectamine™ 3000 Reagent Protocol
USER GUIDE
Document Part No. 100022234 Publication No. MAN0009872 Rev C.0
Package
Contents
Storage
Conditions
Required
Materials
Timing
Selection
Guide
Catalog Numbers
L3000001
L3000008
L3000015
L3000075
L3000150
∤
∤∤
∤
Size:
0.1 mL
0.75 mL
1.5 mL
5 × 1.5 mL
15 mL
Store at 4°C (do not freeze).
Plasmid DNA (0.5–5 μg/μL stock)
Opti-MEM™ Reduced Serum Medium
Microcentrifuge tubes
Preparation: 10 minutes
Incubation: 10–15 minutes
Final Incubation: 1–3 days
Lipofectamine™ Reagents
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Protocol Outline
A. Plate cells so they will be 70–90% confluent at the time of transfection.
B. Prepare plasmid DNA-lipid complexes (recommend 2 doses of lipid).
C. Add DNA-lipid complexes to cells.
Transfection Amounts
Component
DNA per well
P3000™ Reagent per well
Lipofectamine™ 3000 Reagent
per well
∤
Important Licensing Information
For Research Use Only. Not for use in diagnostic procedures.
6-well
500 ng
2500 ng
0.2 μL
1 μL
5 μL
0.15 and
0.3 μL
0.75 and
1.5 μL
3.75 and
7.5 μL
To transfect cells with siRNA, follow the protocol as described for DNA but
do not add P3000™ Reagent when diluting the siRNA (step 3).
Limited Product Warranty
∤
∤
24-well
100 ng
Transfection of siRNA
∤
Lipofectamine™ 3000 Reagent is a proprietary formulation
for transfecting nucleic acids into a wide range of
Product
eukaryotic cells and especially designed for hard to
Description
transfect cells
Make DNA-Lipofectamine™ 3000 complexes in serum-free
medium such as Opti-MEM™ Reduced Serum Medium
and add directly to cells in culture medium, in the
presence or absence of serum/antibiotic.
It is not necessary to remove complexes or change/add
Important
medium after transfection.
Guidelines
The amount of Lipofectamine™ 3000 Reagent for
successful transfection varies. Start any new transfection
by testing the recommended two concentrations of
Lipofectamine™ 3000 Reagent to determine an optimum
amount.
Visit our product page for additional
Online
information and protocols. For support
Resources
visit thermofisher.com/support
96-well
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subsidiaries unless otherwise specified.
10 February 2016
For support visit thermofisher.com/support
Lipofectamine™ 3000 Reagent Protocol
Transfect cells according to the following table. Use the indicated volume of DNA and P3000™ Reagent with each of the two volumes of Lipofectamine™ 3000 (when
performing optimization). Each reaction mix volume is for one well and accounts for pipetting variations. Scale volumes proportionally for additional wells.
Day 0
Timeline
Steps
Seed cells to be 70–90%
confluent at transfection
1
Diluted Lipofectamine™ 3000
2
Vortex 2–3 sec
Diluted DNA
Day 1
3
Prepare master mix of DNA
by diluting DNA in OptiMEM™ Medium, then add
P3000™ Reagent – Mix well
Add Diluted DNA to
each tube of Diluted
Lipofectamine™ 3000
Reagent (1:1 ratio)
4
5
Dilute Lipofectamine™ 3000
Reagent in Opti-MEM™
Medium (2 tubes) – Mix well
10-15
Incubate
Procedure Details (Two Reaction Optimization)
Component
96-well
Adherent cells
1–4 × 10
Opti-MEM™ Medium
Add DNA-lipid complex to
cells
5 µL × 2
25 µL × 2
125 µL × 2
0.15 and 0.3 µL
0.75 and 1.5 µL
3.75 and 7.5 µL
Opti-MEM™ Medium
10 µL
50 µL
250 µL
DNA (0.5–5 µg/µL)
0.2 µg
1 µg
5 µg
P3000™ Reagent (2 µL/µg DNA)
0.4 µL
2 µL
10 µL
Diluted DNA (with P3000™ Reagent)
5 µL
25 µL
125 µL
Diluted Lipofectamine™ 3000 Reagent
5 µL
25 µL
125 µL
Lipofectamine™ 3000 Reagent
96-well
24-well
6-well
DNA-lipid complex
10 µL
50 µL
250 µL
DNA amount
100 ng
500 ng
2500 ng
P3000™ Reagent
0.2 µL
1 µL
5 µL
0.15 and 0.3 µL
0.75 and 1.5 µL
3.75 and 7.5 µL
Lipofectamine 3000 Reagent used
Day 2–4
Visualize/analyze
transfected cells
5
Incubate for 10–15 minutes at room temperature.
™
7
6-well
0.25–1 × 106
Component (per well)
6
24-well
0.5–2 × 10
4
Incubate cells for 2–4 days at 37°C. Then, analyze transfected cells.
For support visit thermofisher.com/support